Identification of an Autoimmune Serum Containing Antibodies Against the Barr Body

Identification of an autoimmune serum containing antibodies against the Barr body

Bo Hong*, Peter Reeves†, Barbara Panning†, Maurice S. Swanson‡§, and Thomas P. Yang*§¶ʈ

*Department of Biochemistry and Molecular Biology, ‡Department of Molecular Genetics and Microbiology, §Center for Mammalian Genetics, and ¶Division of Pediatric Genetics, University of Florida College of Medicine, Gainesville, FL 32610; and †Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94140

Communicated by Stanley M. Gartler, University of Washington, Seattle, WA, May 24, 2001 (received for review April 20, 2001) Transcriptional inactivation of one X chromosome in mammalian components and structure of the Barr body itself. The Barr body female somatic cells leads to condensation of the inactive X has been examined by electron microscopy (6, 7), and the results chromosome into the heterochromatic sex chromatin, or Barr body. indicate the possibility of a special nuclear envelope attachment Little is known about the molecular composition and structure of region for the Barr body. But these studies provide few insights the Barr body or the mechanisms leading to its formation in female into the possible composition or macromolecular organization of nuclei. Because human sera from patients with autoimmune dis- the inactive X chromosome. The Barr body and individual genes eases often contain antibodies against a variety of cellular com- on the inactive X have been probed with nucleases, particularly ponents, we reasoned that some autoimmune sera may contain DNase I, to analyze molecular structure. Nick translation assays antibodies against proteins associated with the Barr body. There- on female cells after fixation and nicking with DNase I have fore, we screened autoimmune sera by immunofluorescence of shown that inactive X chromatin is resistant to nick translation human fibroblasts and identified one serum that immunostained a (6, 8). However, analysis of general DNase I sensitivity of the distinct nuclear structure with a size and nuclear localization X-linked mouse Hprt and human Pgk genes in unfixed cells (9, consistent with the Barr body. The number of these structures was 10) showed a much smaller difference in sensitivity between the consistent with the number of Barr bodies expected in diploid active and inactive alleles (Յ2-fold) than would be expected for female fibroblasts containing two to five X chromosomes. Immu- highly condensed heterochromatin (i.e., inactive X) versus un- nostaining with the serum followed by fluorescence in situ hybrid- condensed euchromatin (i.e., active X). Recent analysis of the ization with a probe against XIST RNA demonstrated that the major three-dimensional organization of the active and inactive X fluorescent signal from the autoantibody colocalized with XIST chromosomes showed the two chromosomes occupy the same RNA. Further analysis of the serum showed that it stains human volume, but the active X appeared flatter with a larger and metaphase chromosomes and a nuclear structure consistent with fuzzier surface than the inactive X, which appeared rounder in the inactive X in female mouse fibroblasts. However, it does not shape with smoother surface structure (11). Two studies have exhibit localization to a Barr body-like structure in female mouse also examined the potential association of the two telomeres and embryonic stem cells or in cells from female mouse E7.5 embryos. resulting loop structure of the inactive X chromosome (12, 13). The lack of staining of the inactive X in cells from female E7.5 Currently, three macromolecules have been shown to colo- embryos suggests the antigen(s) may be involved in X inactivation calize with the Barr body or inactive X chromosome. at a stage subsequent to initiation of X inactivation. This demon- Perichromin, a nuclear envelope protein directly or indirectly stration of an autoantibody recognizing an antigen(s) associated bound to DNA (14), has been reported to be associated with the with the Barr body presents a strategy for identifying molecular Barr body (15). However, its role in X inactivation, if any, is components of the Barr body and examining the molecular basis of unknown. The XIST gene encodes a large nuclear RNA found X inactivation. to be associated exclusively with the inactive X chromosome by fluorescence in situ hybridization (FISH) (16, 17). This RNA uring early mammalian female embryogenesis, one of the exhibits no conserved and extended ORF (16, 18), is transcribed Dtwo transcriptionally active X chromosomes is inactivated in only from the inactive X chromosome (19, 20), and is essential each cell of the embryo (1). The stable inactivation of genes on for normal X inactivation (21, 22). The mechanisms by which the one of the two X chromosomes in females functionally equalizes XIST gene and RNA function in X inactivation are currently the apparent dosage imbalance of X-linked genes between males unresolved. The histone variant macroH2A1.2 also is reported to and females. This chromosome-wide transcriptional silencing is be concentrated on the inactive X chromosome (23, 24), al- associated with condensation of the inactive X chromosome into though association of macroH2A1.2 with the inactive X does not the heterochromatic sex chromatin or Barr body, a unique appear to be required for either initiating or maintaining tran- constituent of the female nucleus identified half a century ago scriptional repression of genes on the inactive X chromosome et al. (2). The Barr body in female interphase nuclei is characteristi- (25, 26). Recently, Perche have reported that, in addition to macroH2A, the core histones H2B and H3 also show pref- cally found as a darkly staining nuclear inclusion commonly erential localization to the Barr body, suggesting that the Barr associated with the nuclear membrane (2). In cells carrying a body may contain a higher density of nucleosomes (27). Con- diploid complement of autosomes, X inactivation and Barr body versely, the inactive X chromosome is reported to be deficient in formation occurs according to the N-1 rule: cells maintain a another novel histone variant, termed H2A-Bbd (28). Despite single active X chromosome and inactivate and condense all these promising findings, knowledge of the composition and remaining X chromosomes (3, 4). However, Barr body formation molecular structure of the Barr body, as well as mechanisms of does not appear to be a requirement for maintaining transcrip- global silencing of genes on the inactive X, remains incomplete. tional repression of genes on the inactive X because rodent– human somatic cell hybrids containing an inactive human X chromosome do not form Barr bodies but continue to maintain Abbreviations: FISH, fluorescence in situ hybridization; ES, embryonic stem. transcriptional silencing of genes on the inactive human X (5). ʈTo whom reprint requests should be addressed. E-mail: [email protected]fl.edu.

The molecular mechanisms for establishing and maintaining The publication costs of this article were defrayed in part by page charge payment. This GENETICS this unique system of differential gene regulation are not well article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. understood, and currently little is known about the molecular §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.151259598 PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8703–8708 Downloaded by guest on September 28, 2021 Fig. 1. Detection of Barr bodies by autoimmune serum 154. Indirect immunoﬂuorescence was performed on diploid human ﬁbroblasts containing different numbers of X chromosomes using autoimmune serum 154 (1:200 dilution) and an FITC-conjugated secondary antibody against human immunoglobulins. Images were collected with a Bio-Rad 1024 ES confocal microscope. (A) GM00468 (46,XY). (B) GM06111 (46,XX). (C) GM00254 (47,XXX). (D) GM01415E (48,XXXX). (E) GM05009C (49,XXXXX). (F) Human-hamster hybrid cell line 8121 (containing an inactive human X chromosome). Arrows indicate Barr bodies.

Antisera from human patients with autoimmune diseases have bryonic fibroblasts in medium containing leukemia inhibitory been used extensively as a tool for studying intracellular struc- factor. Female mouse fibroblasts containing three X chromo- ture and function (29, 30). We reasoned that a small subpopu- somes were a gift of Catherine Brisken (Whitehead Institute). lation of autoimmune patients may carry antibodies against one or more components of the Barr body. Further, autoantisera Isolation of Mouse Embryos. Cells from E7.5 embryos were isolated with antibodies against the Barr body then could be used to as described (32). identify the corresponding Barr body-associated antigen. There- fore, to examine whether or not human autoimmune sera may be Indirect Immunofluorescence. Human fibroblast cells were grown useful as probes of Barr body composition and structure, we overnight on glass microscope slides and fixed with 2% form- screened samples of autoantisera by an indirect immunofluo- aldehyde for 15 min at room temperature. Fixed cells were rescence assay on male and female fibroblasts and examined the treated with acetone at Ϫ20°C for 5 min, then at room temper- ͞ immunostaining patterns for a female-specific staining pattern ature blocked with 3% BSA in PBS (2 mM KH2PO4 8mM ͞ ͞ and colocalization of antibody with the Barr body. Screening of Na2HPO4 2.5 mM KCl 140 mM NaCl, pH 7.2) for 15 min, 255 different autoimmune sera identified one serum containing incubated with diluted autoantiserum for 1 h, washed with PBS, antibodies against the Barr body in both human and mouse incubated with FITC-conjugated goat anti-human Ig for 1 h, fibroblasts. washed again with PBS, counterstained with 1 ␮g͞ml DAPI in PBS for 10 min, mounted in 1 mg/ml p-phenylenediamine͞10 Materials and Methods mM Tris⅐HCl, pH 8.5͞90% glycerol, and examined with an Autoimmune Sera. Sera were obtained from patients with auto- Olympus fluorescence microscope. Images in Figs. 1 and 3 were immune diseases including systemic lupus erythematosus, sclero- collected with a Bio-Rad 1024-ES confocal microscope. derma, and mixed connective tissue disease. Each serum sample was assayed at dilutions of 1:20 and 1:200. Immunostaining Followed by RNA FISH. ES cells and trypsinized embryos were affixed to glass slides by using a cytospin appa- Cells and Cell Culture. The human fibroblast cell lines GM 06111 ratus. Fibroblasts were grown directly on glass slides. Immuno- (46,XX), GM 00254 (47,XXX), GM01415E (48,XXXX), staining was performed first as described above, except that yeast GM05009C (49,XXXXX), and GM00468 (46,XY) were pur- tRNA (0.5 mg͞ml) and RNasin (0.4 units͞␮l, Promega) were chased from the NIGMS Human Genetic Mutant Cell Repos- included at every step to prevent RNA degradation. After itory and grown according to the recommended conditions. immunostaining, the labeled secondary antibody was fixed with Human–hamster hybrid cell line 8121 contains an inactive 2% formaldehyde in PBS, and the slides were dehydrated human X chromosome in a rodent cell background (31). Male through an ethanol series from 70 to 100%. Double-stranded and female mouse embryonic stem (ES) cell lines J1 and 2-1, DNA probes were indirectly labeled by incorporation of biotin- respectively, were grown on mitotically inactivated mouse em- conjugated dCTP by random priming (GIBCO͞BRL) reactions

8704 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.151259598 Hong et al. Downloaded by guest on September 28, 2021 using the plasmid pXist3K for mouse Xist (33) and pG1A (17) antibodies against the Barr body. Initial screening of 255 auto- as templates. Labeled probes were precipitated with yeast tRNA, immune sera at two different dilutions showed that a majority of mouse COT-1 DNA, and sheared salmon sperm DNA, then the sera contained autoantibodies against nuclear antigens, but dried and resuspended in hybridization solution (Hybridsol VII, staining of cytoplasmic antigens also was commonly observed Oncor). Hybridizations were carried out at 37°C overnight in a (data not shown). One antiserum, no. 154, exhibited an immu- humidified chamber. Slides were washed, and probe hybridiza- nofluorescence pattern by optical microscopy consistent with tion was detected with Cy3 conjugated avidin (Amersham Phar- staining of the Barr body where 47,XXX female fibroblasts macia), stained with DAPI, and mounted in antifade mounting showed a distinctly more intense staining of two nuclear struc- media (Vectashield, Vector Laboratories) as described (34). tures similar in size and location to Barr bodies, and 46,XY male fibroblasts lacked this staining pattern. Nearly every female Preparation of Mitotic Cells. The method used was modified from nucleus that showed staining of Barr body-like structures by Jeppesen et al. (35). Cells in log-phase were treated with DAPI showed immunostaining of the same Barr body-like ␮ ͞ colcemid (0.05 g ml) for 4 h, trypsinized, and washed twice structures by serum 154. with PBS. The cells were swollen in 0.075 M KCl supplemented To further examine the possibility of Barr body staining, ͞ ␮ with a protease inhibitor mixture (1 mM benzamide 0.5 g/ml serum 154 was used in indirect immunofluorescence assays of ͞ ␮ leupeptin 1.5 M bestatin in H2O; 0.5 mM phenylmethylsulfo- human fibroblasts carrying one to five X chromosomes and ͞ ␮ ͞ ␮ nyl fluoride 1.5 M pepstatin A 1 g/ml chymostatin solublized examined by confocal microscopy (Fig. 1). The number of in dimethyl sulfoxide) at room temperature for 15 min and spun intensely fluorescent nuclear structures stained by serum 154 onto a glass slide with a Shandon cytospin at 700 rpm at room was equal to that expected from the N-1 rule for Barr body temperature for 10 min. Slides were then incubated in KCM ͞ ͞ ⅐ ͞ formation (Fig. 1 A–E). Male fibroblasts exhibited no staining of buffer (120 mM KCl 20 mM NaCl 10 mM Tris HCl, pH 7.5 0.5 a Barr body-like nuclear structure (Fig. 1A), XX female fibro- mM EDTA) supplemented with the protease inhibitor mixture blasts showed immunofluorescent staining of a single Barr at room temperature for 10 min, fixed with 2% formaldehyde at Ϫ body-like nuclear structure (Fig. 1B), XXX female fibroblasts room temperature for 15 min, treated with acetone at 20°C for showed staining of two Barr body-like structures (Fig. 1C), etc. 3 min, and used for indirect immunofluorescence. The absence of a highly localized Barr body-like staining pattern in cell line 8121 (Fig. 1F), a rodent–human hybrid cell line Western Analysis. Total cellular proteins were prepared as de- containing an inactive human X chromosome that does not form scribed (36). Nuclear proteins were isolated according to Ped- a Barr body (5), further supports the notion that serum 154 erson (37) with modifications. Briefly, cells were incubated at contains antibodies against the Barr body. room temperature in TM-2 buffer (0.01 M Tris, pH 7.4͞0.002 M To confirm that serum 154 stains the Barr body, we performed MgCl ) supplemented with the protease inhibitor mixture (see 2 colocalization studies with XIST RNA by FISH. Previous studies above) for 10 min, treated with 0.5% (vol͞vol) Triton X-100 in have shown that XIST RNA accumulates over the inactive X TM-2 buffer at room temperature for 10 min, and lysed with a chromosome and ‘‘paints’’ the inactive X by FISH (17). There- Dounce homogenizer. Nuclei were collected by spinning at fore, human fibroblasts (47,XXX) were first stained by indirect 600 ϫ g at 4°C for 10 min and washed twice with TM-2 buffer. immunofluorescence with serum 154, then subjected to FISH Nuclear proteins were extracted by lysing the nuclei in sample XIST buffer (4% SDS͞83 mM Tris base͞127 mM Tris⅐HCl, pH 8.0). with an DNA probe. As shown in Fig. 2, fluorescent signals The lysates were sonicated and fractionated by SDS͞ for serum 154 (Fig. 2B) and the XIST probe (Fig. 2C) colocalized polyacrylamide gels. (Fig. 2D) to nuclear structures consistent in size, location, and Western blotting was performed according to standard meth- number with Barr bodies, as confirmed with DAPI staining (Fig. ods (36). Proteins were fractionated on 10.5% SDS͞ 2A). These studies confirm that our screening of 255 human polyacrylamide gels and transferred to a nitrocellulose mem- autoimmune sera identified one sample that contains antibodies brane. After blocking with 5% nonfat milk plus 1% Tween-20, recognizing one or more components of the Barr body. the membrane was incubated with serum 154 (diluted 1:4,000). Signals were detected with a horseradish peroxidase–anti- Analysis of Metaphase Chromosomes. To determine whether the human IgG (1:35,000, Promega) and ECL reagents (Promega). Barr body-associated antigen(s) recognized by serum 154 remains associated specifically with the inactive X chromosome at Results metaphase, metaphase chromosome spreads of 47,XXX female Screening of Autoantisera. Because antisera from patients with fibroblasts were analyzed by indirect immunofluorescence. As autoimmune diseases often contain antibodies against a variety shown by representative metaphase spreads in Fig. 3, the au- of intracellular components (29, 30, 38), we examined the toantiserum showed strong immunostaining of all chromosomes possibility that sera from certain autoimmune patients might in both female (Fig. 3A) and male (Fig. 3B) cells, with some contain antibodies against the Barr body. Antisera from 255 chromosomal regions showing slightly more intense staining autoimmune patients with systemic lupus erythematosus, sclero- than others. There does not appear to be preferential staining of derma, or mixed connective tissue disease were screened by centromeric heterochromatin in metaphase chromosomes. A indirect immunofluorescence for staining of the Barr body. similar pattern of immunostaining also was observed with meta- Cultured male (GM00468; 46,XY) and female (GM00254; phase chromosomes from female mouse embryo fibroblasts 47,XXX) fibroblasts were assayed pairwise with each autoim- (data not shown). These data suggest that the antigen(s) recog- mune serum at 1:20 and 1:200 dilutions of serum and examined nized by serum 154 is not preferentially localized only to the by fluorescence light microscopy. Over 100 cells from multiple inactive X chromosome at metaphase but is present on all fields and at different magnifications were examined for each metaphase chromosomes. This further suggests that the antiserum dilution. Each serum sample was examined for a fluo- gen(s) is unlikely to play a role unique to X inactivation. rescent staining pattern consistent with binding to the Barr body. Nonetheless, at interphase, the antigen(s) is preferentially con- Because all X chromosomes in excess of one per diploid genome centrated over the inactive X (see Fig. 1). The staining pattern are inactivated (the N-1 rule) (3, 4), 47,XXX female cells should of metaphase chromosomes with serum 154 is in contrast to the

contain two preferentially stained nuclear structures frequently pattern seen with antibodies to macroH2A1.2, which preferen- GENETICS associated with the nuclear membrane, and male cells should not tially stained only the inactive X chromosome in metaphase show any staining of such structures if an antiserum contains spreads (23).

Hong et al. PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8705 Downloaded by guest on September 28, 2021 single intensely stained nuclear structure with a size and location consistent with staining of the inactive X chromosome (data not shown). XY male fibroblast nuclei (Fig. 4A) showed no evidence of a localized accumulation of antibody similar to that seen in XX or XXX female nuclei. These results strongly suggest serum 154 crossreacts with the inactive X chromosome in mouse cells and that the inactive X chromosome-associated epitope(s) recognized by the autoimmune serum appears to be evolutionarily conserved in humans and mice. This is consistent with the observation that both human and mouse metaphase chromosomes are also immunostained by the autoimmune serum 154 (see above). Undifferentiated male and female mouse ES cells were also subjected to staining by indirect immunofluorescence with serum 154 to examine the immunostaining pattern before the onset of X inactivation. Undifferentiated female ES cells retain two active X chromosomes and, therefore, do not form a Barr body in interphase cells. Male and female ES cells were first immunostained with serum 154, then subjected to FISH with a probe for Xist RNA. As shown in Fig. 4 C and D, both male and female ES cells exhibited a very diffuse immunostaining pattern in the nucleus with no highly localized accumulation of fluorescence at one or more discrete sites. There appeared to be no preferential colocalization of the antigen(s) with Xist RNA or accumulation of the antigen(s) over either of the X chromosomes in female ES cells before the initiation of X inactivation (Fig. 4D). These results are consistent with the notion that the major antigen(s) Fig. 2. Colocalization of XIST RNA and major sites of immunostaining by autoimmune serum 154. Indirect immunofluorescence with autoimmune se- recognized by serum 154 is associated with formation of a Barr rum 154 (1:200) followed by RNA FISH with a probe against the XIST RNA were body. There also appeared to be no preferential staining of a performed on human female fibroblast cells GM00254 (47,XXX). (A) DAPI structure suggestive of a macrochromatin body (more recently staining. (B) Immunostaining with serum 154. (C) FISH with probe for XIST identified as the centrosome), an intracellular structure stained RNA. (D) The merged images of B and C. in both undifferentiated male and female ES cells by antibodies against histone macroH2A1.2 (24, 25). This further indicates that the Barr body-associated antigen recognized by serum 154 is Analysis of Mouse Fibroblasts, ES Cells, and Embryos. As shown in unlikely to be macroH2A1.2. Fig. 4, we also examined the immunostaining pattern of serum We also examined the staining pattern of serum 154 in cells 154 in female mouse fibroblasts, ES cells, and E7.5 embryos. Fig. from male and female mouse E7.5 embryos. At this stage, most 4 A and B show indirect immunofluorescence of XY male and cells of female embryos have just undergone initiation of X XXX female mouse fibroblasts, respectively, stained with serum inactivation as suggested by the large proportion (80–90%) of 154, then subjected to FISH with a probe for Xist RNA. The cells that exhibit high-level monoallelic association of Xist RNA XXX female nuclei showed an accumulation of immunofluo- with the inactive X. The remaining cells exhibit a differential rescence at two sites in three of four nuclei, sites that also biallelic pattern of Xist RNA association (i.e., a site of low-level colocalized with Xist RNA. These data are consistent with Xist RNA accumulation associated with the eventual active X immunostaining of the mouse inactive X chromosome. Exami- chromosome, and a site of high level Xist RNA accumulation nation of multiple preparations and fields (Ն100 nuclei) indi- associated with the eventual inactive X chromosome) believed to cated Ϸ75% of XXX mouse fibroblasts showed intense staining indicate cells that are undergoing initiation of X inactivation (32, 39). Cells from trypsinized male and female E7.5 embryos were of two nuclear bodies. Similar indirect immunofluorescence stained by indirect immunofluorescence with serum 154 fol- experiments on normal XX female mouse fibroblasts showed a lowed by FISH with a probe for Xist RNA. As shown in Fig. 4 E and F, nuclei from female (and male) E7.5 embryos demonstrated a highly diffuse immunostaining pattern over the entire nucleus with serum 154 and did not exhibit an immunostaining pattern suggestive of localization to the Barr body (or to a macrochromatin body). There was clearly no evidence for colocalization of discrete sites of antibody accumulation with sites of Xist RNA accumulation. Furthermore, close examination of the panel in Fig. 4F of XX E7.5 embryo cells showed one nucleus (on the right) with a differential biallelic pattern of Xist RNA localization (both a weak and a strong site of Xist accumulation) suggestive of a cell in the process of X inactivation. This nucleus did not demonstrate discrete localization of autoantibody staining with either site of Xist RNA expression and accumulation (i.e., the active or inactive X chromosomes), indicating that the Fig. 3. Immunostaining of mitotic chromosomes with autoimmune serum autoimmune serum does not recognize an epitope that accumu- 154. Metaphase chromosomes were prepared from human fibroblast cells. (A) lates on the inactive X chromosome at the same time as Xist GM00254 (47,XXX). (B) GM00468 (46,XY). The chromosomal staining patterns RNA. Taken together, these data would suggest that the anti- shown are representative of multiple fields and preparations from each cell gen(s) recognized by serum 154 is unlikely to be involved in the line. initiation of X inactivation.

8706 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.151259598 Hong et al. Downloaded by guest on September 28, 2021 Fig. 4. Analysis of mouse cells with autoimmune serum 154. Immunostaining with autoimmune serum 154 and Xist FISH were performed on the following. (A) XY male fibroblasts. (B) XXX female fibroblasts. (C) XY male ES cells. (D) XX female ES cells. (E) Cells from XY male E7.5 embryos. (F) Cells from XX female E7.5 embryos. The staining patterns shown are representative of multiple preparations and fields for each cell type.

Western Blot Analysis. To determine the range of cellular proteins on both male and female metaphase chromosomes (see Fig. 3). that react with serum 154 and detect the major proteins recog- Nonetheless, these polypeptides appear to be specifically re- nized by the autoimmune serum, Western blot analysis was cruited to the Barr body in female cells at interphase (given the performed on total cellular and nuclear proteins from male and intense immunofluorescence signal at the Barr body relative to female fibroblasts (see Fig. 5). The autoimmune serum recog- background staining) and are likely to be integral components of nized a single major 39-kDa polypeptide in human extracts, the interphase Barr body, possibly functioning in the condensa- which was significantly enriched in the nuclear fraction. This tion of chromatin of the inactive X (see Discussion). The size of 39-kDa band was present at apparently similar levels in both the major polypeptides recognized by the autoimmune serum male and female extracts, indicating this major antigen is not argues against the possibility that the Barr body-associated female-specific. Two polypeptides at 39 and 41 kDa in mouse immunofluorescence signal from serum 154 could be because of extracts also were recognized by serum 154. These murine core histones (27). polypeptides were again enriched in the nuclear fraction and Discussion present in both female and male cells at similar levels. If these We have identified a human autoimmune serum that immunos- major polypeptides represent the Barr body-associated antigens tains a nuclear structure in female human and mouse fibroblasts recognized by serum 154, they are constituents of the nucleus in that is consistent in size, location, and expected number with the both male and female cells and unlikely to function only in X Barr body. Furthermore, the immunostained structure colocal- inactivation. This is supported by the observation that the izes with XIST͞Xist RNA in both human and mouse fibroblasts, antigen(s) recognized by the autoimmune serum is also present demonstrating its association with the inactive X chromosome. The antigen(s) recognized by this serum shows features different from the known Barr body-associated factors, perichromin, XIST͞Xist RNA, and histone macroH2A1.2. The staining pattern of serum 154 does not show strong immunofluorescence at the nuclear periphery, where perichromin is preferentially localized (14), suggesting the antigen to serum 154 is not perichromin. The autoimmune serum also does not colocalize with Xist RNA in cells from female E7.5 embryos, suggesting that the antigen(s) recognized by the autoimmune serum is not associated with Xist RNA at, or shortly after, the time X inactivation is initiated. Although macro H2A1.2 and the major antigen recognized by serum 154 have similar molecular weights (as suggested by the Western blot in Fig. 5), their immunolo- calization on metaphase chromosomes and in mouse ES cells exhibit distinctly different patterns. Antibodies against macro H2A1.2 show preferential immunostaining of the inactive X chromosome in metaphase chromosomes, whereas serum 154 strongly stains all metaphase chromosomes (see Fig. 3). In undifferentiated ES cells, antibodies against macro H2A1.2 show localization to a distinct macrochromatin body in both male and Fig. 5. Western analysis of human and mouse ﬁbroblasts with autoimmune female ES cells, whereas serum 154 shows no highly localized serum 154. Nuclear (N) or whole cell (T) extracts were prepared from male or staining suggestive of a macrochromatin body in these cells (see

female ﬁbroblasts and subjected to Western blot analysis by using autoim- Fig. 4). Serum 154 also does not show staining of a macrochro- GENETICS mune serum 154. Molecular weights were estimated from molecular weight matin body in cells from E7.5 embryos (Fig. 4). Furthermore, we standards run in adjacent lanes (not shown) and are indicated as labeled. also have expressed a macroH2A1.2-GFP (green fluorescent

Hong et al. PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8707 Downloaded by guest on September 28, 2021 protein) fusion protein in 293 cells, and Western blot analysis of corresponding antigen(s) plays a role unique to formation of these recombinant cells using serum 154 does not show a notable heterochromatin. band at the expected size of the fusion protein (B.H., B.P., and At present, we do not know the identity, age, sex, or disease T.P.Y., unpublished data). These results indicate the Barr body- state of the patient who provided serum 154. However, we have associated antigen recognized by serum 154 is unlikely to be just completed a screening of another independent collection of macroH2A1.2. Furthermore, the molecular weights of the major 185 autoantisera and identified a second serum sample that bands seen in Western blots of human and mouse cell extracts by immunostains the Barr body in human female fibroblast cells at using serum 154 (Fig. 5) are significantly larger than each of the very high frequency with strong signal intensity (W. H. Brooks, core histones, arguing against the possibility that serum 154 M. Sato, W. H. Reeves, and T.P.Y., unpublished data). This recognizes any of the core histones that are reported to be newly identified serum sample is derived from a female patient concentrated at the Barr body (27). In addition, the antigen(s) with systemic lupus erythematosus. recognized by serum 154 is not acid extractable (data not Our identification of autoimmune sera with antibodies against shown), further evidence that serum 154 is unlikely to recognize the Barr body indicates that antigens associated with the Barr a histone family member. body are capable of eliciting an autoimmune response. This leads ͞ Because serum 154 shows colocalization with XIST Xist RNA to the possibility that sera from a distinct subpopulation of in female fibroblast cells (Figs. 2 and 4B), but not in cells from autoimmune patients (Ϸ0.5%) may contain antibodies against a female E7.5 embryos (Fig. 4F) when cells are undergoing or just variety of Barr body-associated antigens. Thus, these autoan- completing initiation of X inactivation (32), the Barr body- tisera may offer an experimental approach for examining the associated antigen(s) recognized by the antiserum is more likely molecular basis for Barr body formation and͞or establishing and to be involved in the process of X inactivation, if at all, at a stage maintaining X chromosome inactivation by providing tools for subsequent to the initiation of inactivation. This might include the identification and characterization of molecular components potential roles in the global stabilization of the inactive state ͞ of the Barr body. We currently are screening phage expression after X inactivation has been established and or maturation and libraries with serum 154 to identify the Barr body-associated condensation of the inactive X into the heterochromatic Barr antigen. body during female development. A role for the antigen(s) in ͞ chromatin chromosome condensation is suggested by the strong We thank Ralph Williams and Robert Eisenberg for kindly providing staining of metaphase chromosomes by the autoantiserum (see autoimmune sera. We also thank Daniel Driscoll for use of his fluores- Fig. 3). However, because the autoimmune serum does not cence microscope, and Wesley Brooks for assistance in preparation of exhibit preferential staining of centromeric heterochromatin in figures. This work was supported by National Institutes of Health Grant metaphase chromosomes (see Fig. 3), it is unlikely that the RO1 GM44286 (to T.P.Y.) and the Sandler Family Foundation (to B.P.).

1. Heard, E., Clerc, P. & Avner, P. (1997) Annu. Rev. Genet. 31, 571–610. 21. Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S. & Brockdorff, N. (1996) 2. Barr, M. L. & Bertram, E. G. (1949) Nature (London) 163, 676–677. Nature (London) 379, 131–137. 3. Lyon, M. F. (1996) Nature (London) 379, 116–117. 22. Marahrens, Y., Panning, B., Dausman, J., Strauss, W. & Jaenisch, R. (1997) 4. Lee, J. T. & Jaenisch, R. (1997) Nature (London) 386, 275–279. Genes Dev. 11, 156–166. 5. Hansen, R. S., Canfield, T. K., Stanek, A. M., Keitges, E. A. & Gartler, S. M. 23. Costanzi, C. & Pehrson, J. R. (1998) Nature (London) 393, 599–601. (1998) Proc. Natl. Acad. Sci. USA 95, 5133–5138. 24. Rasmussen, T. P., Mastrangelo, M., Eden, A., Pehrson, J. R. & Jaenisch, R. 6. Dyer, K. A., Riley, D. & Gartler, S. M. (1985) Chromosoma 92, 209–213. (2000) J. Cell Biol. 150, 1189–1198. 7. Wolstenholme, D. R. (1965) Chromosoma 16, 453–462. 25. Mermoud, J. E., Costanzi, C., Pehrson, J. R. & Brockdorff, N. (1999) J. Cell 8. Kerem, B. S., Goitein, R., Richler, C., Marcus, M. & Cedar, H. (1983) Nature Biol. 147, 1399–1408. (London) 304, 88–90. 26. Csankovszki, G., Panning, B., Bates, B., Pehrson, J. R. & Jaenisch, R. (1999) 9. Yang, T. P. & Caskey, C. T. (1987) Mol. Cell. Biol. 7, 2994–2998. Nat. Genet. 22, 323–324. 10. Riley, D. E., Goldman, M. A. & Gartler, S. M. (1986) Somatic Cell Mol. Genet. 27. Perche, P., Vourc’h, C., Konecny, L., Souchier, C., Robert-Nicoud, M., 12, 73–80. Dimitrov, S. & Khochbin, S. (2000) Curr. Biol. 10, 1531–1534. 11. Eils, R., Dietzel, S., Bertin, E., Schrock, E., Speicher, M. R., Ried, T., 28. Chadwick, B. P. & Willard, H. F. (2001) J. Cell Biol. 152, 375–384. Robert-Nicoud, M., Cremer, C. & Cremer, T. (1996) J. Cell Biol. 135, 29. Tan, E. M. (1989) Adv. Immunol. 44, 93–151. 1427–1440. 30. Lerner, M. R. & Steitz, J. A. (1979) Proc. Natl. Acad. Sci. USA 76, 5495–5497. 12. Walker, C. L., Cargile, C. B., Floy, K. M., Delannoy, M. & Migeon, B. R. (1991) 31. Ledbetter, S. A., Schwartz, C. E., Davies, K. E. & Ledbetter, D. H. (1991) Am. J. Proc. Natl. Acad. Sci. USA 88, 6191–6195. Med. Genet. 38, 418–420. 13. Dietzel, S., Eils, R., Satzler, K., Bornfleth, H., Jauch, A., Cremer, C. & Cremer, 32. Panning, B., Dausman, J. & Jaenisch, R. (1997) Cell 90, 907–916. T. (1998) Exp. Cell Res. 240, 187–196. 33. Panning, B. & Jaenisch, R. (1996) Genes Dev. 10, 1991–2002. 14. McKeon, F. D., Tuffanelli, D. L., Kobayashi, S. & Kirschner, M. W. (1984) Cell 34. Johnson, C. V., Singer, R. H. & Lawrence, J. B. (1991) Methods Cell Biol. 35, 36, 83–92. 73–99. 15. Gartler, S. M., Dyer, K. A. & Goldman, M. A. (1992) Mol. Genet. Med. 2, 35. Jeppesen, P., Mitchell, A., Turner, B. & Perry, P. (1992) Chromosoma 101, 121–160. 322–332. 16. Brown, C. J., Hendrich, B. D., Rupert, J. L., Lafreniere, R. G., Xing, Y., 36. Gallagher, S. R. (1999) in Current Protocols in Molecular Biology, eds. Ausubel, Lawrence, J. & Willard, H. F. (1992) Cell 71, 527–542. F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. 17. Clemson, C. M., McNeil, J. A., Willard, H. F. & Lawrence, J. B. (1996) J. Cell & Struhl, K. (Wiley, New York), Vol. 2, pp. 10.2A.1–10.2A.34. Biol. 132, 259–275. 37. Pederson, T. (1998) in Cell: A Laboratory Manual, eds. Spector, D. L., 18. Brockdorff, N., Ashworth, A., Kay, G. F., McCabe, V. M., Norris, D. P., Goldman, R. D. & Leinwand, L. A. (Cold Spring Harbor Lab. Press, Plainview, Cooper, P. J., Swift, S. & Rastan, S. (1992) Cell 71, 515–526. NY), Vol. 1, p. 43.1. 19. Brockdorff, N., Ashworth, A., Kay, G. F., Cooper, P., Smith, S., McCabe, V. M., 38. Hollingsworth, P. N., Pummer, S. C. & Dawkins, R. L. (1996) in Autoantibodies, Norris, D. P., Penny, G. D., Patel, D. & Rastan, S. (1991) Nature (London) 351, eds. Peter, J. B. & Shoenfeld, Y. (Elsevier, New York), pp. 74–90. 329–331. 39. Sheardown, S. A., Duthie, S. M., Johnston, C. M., Newall, A. E., Formstone, 20. Brown, C. J., Ballabio, A., Rupert, J. L., Lafreniere, R. G., Grompe, M., E. J., Arkell, R. M., Nesterova, T. B., Alghisi, G. C., Rastan, S. & Brockdorff, Tonlorenzi, R. & Willard, H. F. (1991) Nature (London) 349, 38–44. N. (1997) Cell 91, 99–107.

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